Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.
Compounds that fluoresce have many uses and are known to be particularly suitable for biological applications where fluorescence is intrinsically more sensitive than absorption as the incidence and observed wavelengths are different. Fluorescence can be used for the detection of whole cells, cellular components, and cellular functions. For example, many diagnostic and analytical techniques require the samples to be fluorescently tagged so that they can be detected. This is achieved by using fluorescent dyes or probes which interact with a wide variety of materials such as cells, tissues, proteins, antibodies, enzymes, drugs, hormones, lipids, nucleotides, nucleic acids, carbohydrates, or natural or synthetic polymers to make fluorescent conjugates.
With synthetic fluorescent probes, ligands are frequently used to confer a specificity for a biochemical reaction that is to be observed and the fluorescent dye provides the means of detect or quantify the interaction. These applications include, among others, the detection of proteins (for example in gels, on surfaces or aqueous solution), cell tracking, the assessment of enzymatic activity and the staining of nucleic acids or other biopolymers.
Long wavelength absorbance usually increases the utility of a fluorescent probe since it reduces the interference from cellular autofluorescence and is less likely to cause photo-damage of labelled biomolecules. Although lasers are particularly useful as a concentrated light source for the excitation of fluorescence, at present the output of powerful lasers is restricted to particular wavelengths of light. Compounds whose excitation spectrum coincide with laser output are therefore of high utility. The argon laser is the most common light source for excitation of fluorescence, and has principal output at 488 nm and a weaker output at 514 nm. Fluorescent compounds that are excited by either of these wavelengths are therefore of particular utility. YAG lasers (532 nm or 473) and HeNe (543 nm, 633 nm) are also becoming common.
Red fluorescent compounds are used extensively in many fields of biological study. Many of these, including Texas red, Tetramethyl rhodamine or red emitting BODIPY dyes require excitation at green wavelengths such as 542 nm. This limits their use in many applications, especially those where the argon-ion laser is used for excitation.
Compounds such as ethidium bromide, can be excited with light from the argon-ion laser, but are not generally suitable for tagging of organic molecules other than nucleic acids. Other compounds such as phycoerythrin, can be excited using the argon-ion laser (488 nm), and emits in the orange wavelengths (ca 580 nm). Phycoerythrin, however, has poor stability and a high molecular weight (ca 240,000 Da) making it unsuitable for many applications such as cell tracking, labelling of nucleic acids or staining proteins.
For staining of proteins, there are a number of methods available. These methods can utilise non-fluorescent compounds, or fluorescent compounds. The most commonly used method utilises Coomassie blue (Bradford assay), which is non-fluorescent. Fluorescence-based protein-detection methods utilise fluorescent dyes, which form a complex with the protein and are intrinsically more sensitive than non-fluorescent methods. Fluorescent staining of proteins has a number of advantages over traditional Silver or Coomassie staining. These advantages include greater sensitivity, lower background interference and greater dynamic range.
For staining of nucleic acids such as DNA and RNA ethidium bromide as a fluorescent stain has been most widely used due to its cost effectiveness and high sensitivity (2 ng/ban of dsDNA). Its uses among researchers have been somewhat limited because it is thought to be carcinogenic. Other fluorescent nucleic acid stains are currently available for quantification of nucleic acids as well as gel staining however in use such stains also have significant disadvantages.
WO01/81351, incorporated herein by reference, describes fluorescent dye compounds based on a furo[3,2-g][2]benzopyran-2,9(9aH)dione core.
Fluorescent dyes are particularly useful in the field of electrophoresis. Electrophoresis allows the separation of charged biomolecules, such as DNA, RNA and/or proteins, by making use of the relative mobilities of the charged molecules in a gel matrix after the application of an electrical field. The distance moved by each molecule in the electrical field depends on the charge, shape and weight of the molecule.
The most commonly used gel matrix for the separation of proteins is polyacrylamide (PAGE electrophoresis). SDS-PAGE is a technique whereby proteins are treated with the anionic detergent sodium dodecyl sulfate (SDS) before electrophoresis. SDS denatures the proteins and coats them with a uniform negative charge. This means that separation is based solely on molecular weight, and SDS-PAGE is typically used to determine the molecular weights of proteins1.
In contrast, nucleic acid bear a single negative charge for every nucleotide (MW approx 500 Daltons) so there is a reasonably constant mass/charge ratio. In the case of nucleic acids it is not necessary to normalise the charge with a detergent.
Whilst a number of fluorescent dyes are known in the art, there is still a need to improve the signal intensity, the signal to background ratio and the sensitivity of fluorescent dyes. There is an additional need to improve the stability of fluorescent complexes formed on an electrophoresis gel matrix.
It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.